Abstract
Frequency of Hepatitis B Core Antibody and Hepatitis B Virus DNA among Apparently Healthy Male Blood Donors in Eastern Libya Mohamed K. Shambesh1,2, Ezzadin A Franka1,2, Amal R. Agila3, Faisal F. Ismail2,3 1Department of Community Medicine, Faculty of Medicine, University of Tripoli, Tripoli, 2National Centre for Disease Control, 3Department of Laboratory, Faculty of Medical Technology, University of Tobruk, Tobruk, Libya Abstract Original Article Background/Objectives: Hepatitis B virus (HBV) infection represents one of the most serious blood transfusion-transmitted viral infections. By implementation of the hepatitis B surface antigen (HBsAg) screening assay, blood banks in Libya have been considerably increased blood transfusion safety in term of protecting against the transmission of HBV infection. However, several studies demonstrated that donors who are HBsAg negative and hepatitis B core antibody (anti-HBc) positive maybe a potential source for posttransfusion hepatitis B. The aim of this study is to determine the presence of anti-HBc and HBV DNA (hepatitis B viral DNA) in healthy HBsAg-negative blood donors in eastern Libya (Tobruk region). Materials and Methods: A total of 500 serum samples were tested for HBsAg and obtained from healthy blood donors in blood bank unit in Tobruk Medical Center. All donors were tested for anti-HBc, using commercial ELISA and microwell methods (MBS-SRL, Milano, Italy). The reactive samples were further tested for the presence of HBV DNA using polymerase chain reaction (PCR). Results: In this study, the seroprevalence rate of anti-HBc sample was 54 (10.8%) among donors. The majority of anti-HBc-positive cases (52 of 54) were in the age group of 20–49 years. Of the 54 anti-HBc-positive samples, 4 (7.4%) were tested positive for HBV DNA by PCR. Conclusion: Among all the samples, the rate of anti-HBc was 10.8%. This finding is comparable to a previous study performed in northwestern Libya. The present study estimated the expected exclusion rate of anti-HBc-positive donated blood that would be an important factor to consider before adopting anti-HBc testing in addition to HBsAg testing as a mandatory screening test to further enhance transfusion safety. Keywords: Blood donors, eastern Libya, hepatitis B virus, hepatitis B core antibody IntRoductIon Hepatitis B virus (HBV) infection remains a global important health problem. It is responsible for a significant number of deaths due to HBV infection-related complications.[1] It is estimated that there are approximately 257 million HBV carriers in the world, of whom 887,000 die annually from HBV infection-related liver diseases.[2] Large efforts have been made in recent decades to prevent incidence of HBV infection. Perhaps, the most important steps have been the implementation of HBV global vaccination programs to all antibody (anti-HBc), and more recently implementing nucleic acid testing (NAT) in many developed countries.[4] In Libya, the carrier state of the disease among the general population was found 2.2%,[5] which considered an area of low-intermediate endemicity (2%–7%) for HBV infection as classified by the WHO.[6] Several preventive measures have been implemented over the past three decades in the country; such as introducing the free mandatory vaccination program in late 1990s to all newborns as well as to high-risk groups; in addition, implementing HBsAg serological testing to newborns and to high-risk groups in several countries, in addition to developing more efficacious treatments to treat Address for correspondence: Dr. Faisal F. Ismail, Department of Laboratory, Faculty of Medical Technology, University of its chronic status,[3] in addition to screening blood and other Tobruk, Tobruk, Libya. blood derivatives for the presence of HBV infection markers E‑mail: faisal.ismail@tu.edu.ly such as hepatitis B surface antigen (HBsAg), hepatitis B core Access this article online Quick Response Code: Website: www.ljmsonline.com DOI: 10.4103/LJMS.LJMS_47_17 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact: reprints@medknow.com How to cite this article: Shambesh MK, Franka EA, Agila AR, Ismail FF. Frequency of Hepatitis B core antibody and Hepatitis B Virus DNA among apparently healthy male blood donors in Eastern Libya. Libyan J Med Sci 2018;2:12-5. 12 © 2018 Libyan Journal of Medical Sciences | Published by Wolters Kluwer - Medknow blood donors in 1980s;[7] in addition, screening for HBsAg is obligatory to all individuals as a part of their preemployment and premarital medical checkup.[8,9] Many studies[10-14] emphasized on the importance of implementing anti-HBc with HBsAg to increase blood safety[15] and showed that a percentage of blood donors who are found negative for HBsAg and positive for anti-HBc may carry circulating hepatitis B viral DNA (HBV DNA) and may be a potential source of posttransfusion hepatitis B, yet blood donation centers in Libya are relying on HBsAg as the only screening assay for detection of HBV infection. In this regard, several studies were performed in the last few years to estimate the percentage of anti-HBc seroprevalence in blood donors in the country and also to estimate the chronic carrier state among blood donors who found to be negative for the presence of HBsAg and positive for anti-HBc by screening anti-HBc blood units using the polymerase chain reaction (PCR) and found that the frequency of anti-HBc in blood donors ranges from 3.9% to 15.6% in different parts of the country.[10-14] This study was conducted to determine the seroprevalence of anti-HBc in healthy HBsAg-negative blood donors in eastern Libya (Tobruk area), to estimate the exclusion rate of the anti-HBc-positive donated blood. This would be an important factor for the health authorities to consider before adopting anti-HBc testing in addition to HBsAg testing as a mandatory screening test to increase transfusion safety until considering introducing an advanced testing system for HBV, such as NAT. mateRIals and methods Study population A total of 500 HBsAg-negative blood samples were prospectively obtained from healthy male blood donors who attended Tobruk hospital blood bank unit between October 22, 2016, and January 30, 2017. All blood donor samples were collected in 5 ml blunt tube and left for 15 min until the serum separated and then centrifuged for 5 min at 3000 RPM. The sera transferred to other tubes and kept frozen at −20 until transferred to National Centre for Disease Control, Tobruk branch. Tobruk city is the capital of Butnan province of Libya, which is located on the Libyan’s eastern Mediterranean coast, sharing around 140 km long Mediterranean coastline, and it has international border with Egypt in the east. Tobruk hospital blood bank unit serves neighboring cities as well as Tobruk. All donors were examined as per blood bank’s standard operating procedure. Ethical consideration The study protocol was approved by the Ethics Committee of the Scientific Research in Tobruk University. All male blood donors were informed about the research, and written consent was obtained from each donor to collect an anonymous serum sample to be used in this study. Donors’ data such as age, address, and donation history were obtained. Donors were classified as replacement donors and voluntary donors; replacement donors who donated blood to their relatives and friends and voluntary donors who voluntarily donated their blood. Serological assays All mandatory viral screening tests for blood-transmitted infections, i.e. HBsAg, anti-HCV, anti-HIV and VDRL for screening of syphilis were performed in the Tobruk Medical Center's blood bank as part of routine mandatory screening tests using commercially available ELISA (Dialab, Microwell method, GmbH) and for the VDRL by hemagglutination assay (Plasmatec Laboratory Products Limited, UK). All blood samples were simultaneously tested for anti-HBc using ELISA microwell methods (MBS-SRL, Milano, Italy). The sera were transferred frozen to the medical laboratory of National Centre for Disease Control, Tobruk, where anti-HBc analysis was performed. All reactive samples were further confirmed in duplicate. Real‑time polymerase chain reaction All 54 anti-HBc-reactive samples were further analyzed by real-time PCR for the presence of HBV viral genome; 500 µL of each sample was extracted and amplified and target HBV genome fragment detected using the COBAS® AmpliPrep/COBAS® TaqMan® HBV Test, version 2.0 system (analysis conducted in France by Taqman Roche, Cerba). The test procedure was carried out according to the manufacturer’s instructions. The sensitivity of the real-time PCR used is 20 IU/mL; the conversion factor is 1 IU = 5.82 copies. Statistical data analysis Data analysis was performed using SPSS computer software (Version 19, SPSS Inc., Chicago, IL, USA). The contributing blood donors were divided into groups according to their age. Chi-square test was used to highlight if there are no statistical significance differences between variables. In all tests, α < 0.05 was regarded statistically significant. All confidence intervals were calculated at the 95% level of statistical significance. Results Libyan Journal of Medical Sciences ¦ Volume 2 ¦ Issue 1 ¦ January-March 2018 13 The study was performed on 500 donor males aged from 17 to 68 years. The donors were divided into age groups, as shown in Figure 1. The donors had not been previously tested for anti-HBc. Of the total 500 male donors, only 182 (36.4%) were voluntary donors who had donated more than once before, and 318 (63.6%) were family or friend replacement donors. All blood samples were negative to other mandatory tests for blood-transmitted infections (HBsAg, anti-HCV, anti-HIV, and syphilis). Of 500 male blood donors, 54 (10.8%) samples were positive results for anti-HBc. The distribution of anti-Hbc antibody positivity was nearly similar among the voluntary donors (n = 20, 10.9%) and the family replacement donors (n = 34, 10.6%). The majority of anti-HBc-positive samples were found in the age group of 20–49 years with a concentration in the age group of 30–39 years (43.2%) [Table 1]. Table 1: Screened age groups by total amount listed anti‑hepatitis B core positive Age groups Anti‑HBc‑positive cases among whole samples Number of donors screened (%) 10-19 0 12 (0.000) 20-29 10 193 (0.052) 30-39 26 192 (0.135) 40-49 16 85 (0.188) >50 2 18 (0.111) Total 54 500 (0.108) Anti-HBc: Hepatitis B core antibody 250 200 150 100 50 0 12 193 192 85 18 < 20 20 - 29 30 - 39 Donors Age Group 40 - 49 > 50 Figure 1: Donors’ number among different age groups Four samples were found to be seropositive for occult HBV infection. This figure represents 0.8% of the whole sample and 7.4% of the anti-HBc-positive samples. The positive PCR samples were found 25, 28, 39, and 54 IU, respectively. All the positive PCR samples were from 30 to >50 years’ age groups, and three cases were from the voluntary donor group and one case from the replacement donor group. The rate of anti-HBc-positive exclusion was estimated that approximately 11 blood units would be excluded from every 100 donated units if anti-HBc testing was to be adopted. Moreover, the study estimates that 8 donated units per 1000 donated units may be potentially infected with HBV. dIscussIon Currently, the only serological screening assay for HBV infection in Libyan blood banks is HBsAg.[5] The present research tested 500 healthy male blood donors in East Libya (Tobruk region) by detecting another serological marker for HBV infection known as total (IgG and IgM) anti-HBc antibodies which was found to be positive 54 (10.8%) blood donors. This percentage is similar to that found in two previous studies conducted by the authors. The first study was performed in 2014 (500 blood donors) from Tripoli region, anti-HBc percentage was found (9.8%),[12] and the second study was performed in 2015 (1256 donors) in the northwestern region of Libya (including Tripoli and its surroundings), anti-HBc frequency was found in 10.5%.[13] However, our finding was considerably low compared with another study done on 200 blood donors in Tripoli Central Hospital blood bank in 2009;[10,11] this difference in “frequency” could be due to the small sample size. In contrast, the rate of anti-HBc in the present study is high compared to that found in a study in 2015 (979 blood donors) in middle northern region of Libya (Misurata and their neighboring cities).[14] This “variation” in anti-HBc rates between the two studies could be related to the fact that there could be small difference in endemic rates of chronic HBV carrier between the two regions,[5] and another possibility is that most of blood donated in Misurata study was classified as family types, which usually had lower levels of HBV infectivity.[14] The prevalence of anti-HBc also has been reported from countries neighboring Libya, Egypt (7.8% and 10.9% in two different studies).[16,17] Although the North African countries show high prevalence in anti-HBc rate, in Europe, the rate of anti-HBc shows lower levels, varying between 0.07% in the UK, 1.5% in Germany,[18] and 4.9% in Italy.[19] In the Middle East, the prevalence of anti-HBc among blood donors has been found to be higher, 17% in Kuwait[20] and 15.3% in Saudi Arabia.[21] In countries elsewhere, the prevalence varies between 6.5% in Iran[22] and a very high prevalence (42%) in Sudan.[23] These variations of anti-HBc levels between different countries were related to the different levels of HBV infections being endemic as classified by the WHO; Libya was classified in the intermediate level of the endemic range (Europe was classified with low levels of being endemic while Asian countries and some African countries were classified with high levels).[6] The frequency of HBV DNA among anti-HBc-positive donors was 7.4%. This frequency is higher than a previous pilot study conducted by the authors in Tripoli region (3%)[10,11] and less than another study performed by the authors in Northern Libya (10.5%).[13] Furthermore, this percentage is higher compared with the DNA-positive sample that found in another study by the same authors in North Middle Libya.[14] In comparison, in this study, the frequency of HBV DNA in anti-HBc-positive donors is low compared with that found in Egypt (11.54%).[16] However, it is roughly similar to that reported in another study in Egypt (6.25%),[17] is higher than that reported in Italy (4.86%),[19] and is also lower than that found in Iran (12.2%).[22] These differences could be related to the endemicity of HBV infection among different countries. This study estimates that about 108 blood donors would be excluded from every 1000 donated persons, if anti-HBc testing were adopted, and approximately 8 donors per 1000 donated persons may possibly be infected with HBV. conclusIon 14 Libyan Journal of Medical Sciences ¦ Volume 2 ¦ Issue 1 ¦ January-March 2018 This study reports a prevalence rate of anti-HBc (10.8%) in eastern region of the Libya, which is comparable to other studies performed in the west of the country and high compared to one study performed in the north middle of the country. The present study estimated that the expected exclusion rate of anti-HBc-positive donated blood which approximately 11 blood donors would be excluded from every 100 donated persons, if anti-HBc testing was to be adopted in addition to HBsAg as a mandatory screening test to further enhance transfusion safety. Furthermore, the implementation of anti-HBc testing would help to find more chronic HBV carriers; this may allow early access to therapy and therefore prevent the complications of HBV infection. Acknowledgment The authors would like to thank all the blood bank staffs in Tobruk Medical Center and all medical laboratory staffs at NCDC, Tobruk, for their assistance in performing serological analysis. A special thanks to the National Authority for Scientific Research (NASR), Libya, for supporting this research. Financial support and sponsorship This work was financially supported by the National Authority for Scientific Research (NASR), Libya. Conflicts of interest There are no conflicts of interest. RefeRences 1. Ly KN, Xing J, Klevens R, Jiles RB, Ward JW, Holmberg SD. The increasing burden of mortality from viral hepatitis in the united states between 1999 and 2007. Annals Intern Med2012;156:271-8. 2. Avalaible from: http://www.who.int/mediacentre/factsheets/fs204/en/ Af. 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Anti-HBc & HBV-DNA detection in blood donors negative for hepatitis B virus surface antigen in reducing risk of transfusion associated HBV infection. Indian J Med Res 2006;123:37-42. 23. Abd El Kader Mahmoud O, Abd El Rahim Ghazal A, El Sayed Metwally D, Elnour AM, Yousif GE. Detection of occult hepatitis B virus infection among blood donors in sudan. J Egypt Public Health Assoc 2013;88:14-8. Libyan Journal of Medical Sciences ¦ Volume 2 ¦ Issue 1 ¦ January-March 2018 15